In the urea biochemical cycle for removal of toxic NG4+, the enzyme arginase catalyzes the hydrolysis of L-arginine to urea and L-ornithine by a mechanism which is not understood at the molecular level. This reaction also provides L-ornithine as biosynthetic precursor to the polyamines, spermine and spermidine, important growth factors. Also through this reaction the intracellular concentration of L-arginine is influenced, thus mediating the arginine-dependent formation of nitric oxide. Nitric oxide is an important neurotransmitter in the brain, a potent cytotoxic agent in macrophages, a vasodilator and the key mediator of penile erection in mammals. Arginase deficiency in the liver leads to hyperammonemia, an inherited disorder leading to neurologic impairment and mental retardation. Several factors have come together recently which offer new hope for understanding the chemical mechanism for arginase's hydrolytic activity. Additionally, an unprecedented redox activity of bacterial catalases. This proposal describes structural and mechanistic studies of the dimanganese active site of arginase. In collaboration with Dr. David Ash from Temple University we propose to use site-directed mutagenesis of protein residues in combination with EPR and Electron Nuclear Double Resonance (ENDOR) spectroscopies to: 1) identify the protein residues which coordinate the two manganese ions at the catalytic site and serve to distinguish arginase from manganese catalases, 2) the products, urea and L-ornithine, 3) determine the functions of both manganese ions in the catalysis of arginine hydrolysis, 4) characterize the chemical mechanism of the newly discovered catalase activity and its potential link to peroxidase activity and synthesis of NO precursors, 5) enhance the catalase activity of arginase by mutagenesis of the manganese ligands, 6) examine the potential arginase activity of a new family of dimanganese complexes which are known to be good structural models for the active site of arginase.